摘要
目的观察细胞凋亡因子核酸内切酶G(endonuclease G,Endo G)基因过表达在镉诱导人胚胎肾细胞HEK-293凋亡中的影响。方法从人肝癌细胞系(human hepatocarcinoma cells,HepG 2)中提取RNA,经逆转录获取互补DNA cDNA),通过聚合酶链反应(PCR)扩增Endo G基因,与pcDNA 3.0相连,构建pcDNA 3.0-Endo G真核表达载体,转染HEK-293细胞,并结合不同浓度氯化镉处理细胞;通过蛋白印迹实验(Western blot)验证基因过表达,吖啶橙/溴乙锭(AO/EB)双染法及流式细胞术(FCM)检测Endo G基因过表达对镉诱导HEK-293细胞凋亡的影响,噻唑蓝法(MTT)检测细胞存活率。结果成功构建pcDNA 3.0-EndoG真核表达载体,Endo G基因扩增片段大小约1 100 bp;该载体转染细胞后,Endo G成功过表达,使HEK-293细胞凋亡率>37%,且随氯化镉浓度增加,Endo G表达量先上升后下降。结论 Endo G以诱导细胞早期凋亡和晚期凋亡为主要形式。
Objective To examine the effects of over-expression of endonuclease G（Endo G） gene on cadmium-induced apoptosis of embryonic kidney cell HEK-293 in vitro.Methods Total RNA was obtained from human hepatocarcinoma cell line（HepG 2） and cDNA was obtained by reverse transcription.The Endo G gene was amplified by PCR.The Endo G was inserted reversely into pcDNA 3.0,and the recombinant plasmid pcDNA 3.0-Endo G was selected and then transferred into DH5α strain.The pcDNA 3.0-Endo G was transfected into HEK-293 cells.Western blot was used to confirm the expression of Endo G gene in HEK-293 cells.Acridine orange/ethidium bromide（AO/EB） and flow cytometry were used to determine the effect of Endo G gene on cadmium-induced apoptosis of HEK-293 cells.The cell proliferation was detected by methyl thiazolyl tetrazolium（MTT） method.Results The size of Endo G gene fragment is about 1100 bp.The eukaryotic expressing plasmid of pcDNA 3.0-Endo G was constructed successfully.The expression of Endo G was first increased and then decreased in HEK-293 cells with the increase of concentration of cadmium chloride.The study with AO/EB and flow cytometry showed that the apoptotic rate of HEK-293 cells over-expressing Endo G gene was over 37% and significantly increased.Conclusion Endo G induces apoptosis of HEK-293 cells with the main form of early and late apoptosis.
出处
《中国公共卫生》
CAS
CSCD
北大核心
2012年第10期1322-1326,共5页
Chinese Journal of Public Health
基金
江苏省高校优势学科建设工程项目
